Bmi-1 promotes the proliferation, migration and invasion, and inhibits cell apoptosis of human retinoblastoma cells via RKIP

Retinoblastoma is one of the most common ocular malignancies in children. Bmi-1, a member of the Polycomb group family of transcriptional repressors, is expressed in a variety of tumors. The purpose of our study was to explore the role of Bmi-1 in retinoblastoma. RT-qPCR and western blot were used for calculating the mRNA and protein levels of Bmi-1 and RKIP. MTT, Wound healing and Transwell assays were performed to measure the proliferation, migration and invasion in retinoblastoma cells. Cell apoptosis was detected by flow cytometry. The volume and mass of transplanted tumors were detected in nude mice. Bmi-1 was over expressed, and RKIP was low expressed in retinoblastoma cells. Bmi-1 promoted cell proliferation, migration and invasion and suppressed cell apoptosis of Y79 and SO-RB50 cells. Downregulation of Bmi-1 and overexpression of RKIP inhibited cell proliferation, migration and invasion, and increased cell apoptosis. The functions of Bmi-1 knockdown on retinoblastoma cells were blocked by RKIP knockdown, but promoted by RKIP. Down-regulated Bmi-1 inhibited xenograft tumor growth, and RKIP exacerbated this inhibitory effect. Bmi-1 served as a potential therapeutic target for improving the efficacy of clinical treatment in retinoblastoma. All the findings revealed the functions of Bmi-1/RKIP axis in retinoblastoma tumorigenesis.


Cell proliferation
The MTT method was used for detection, and the cells in the logarithmic growth phase were used to perform the experiment.A single cell suspension with a cell number of 1 × 10 5 /mL was prepared and seeded in a 96-well plate at 200 μL/well.After 24, 48 and 72 h, cell viability was measured by MTT. 4 h before the end of the experiment, 20 μL of 5 mg/mL MTT was added to each well, and the incubation continued for 4 h.After the experiment, the culture solution was centrifuged and discarded.The microplate reader detects the absorbance of each well at a wavelength of 490 nm.

Transwell assay
Transwell assay was applied to test cell invasion ability at 24 h after transfection.In brief, non-serum RMPI-1640 Medium was used to prepare single cell suspensions at a cell concentration of 3 × 10 4 cells/mL.Before invasion assay, the upper chamber was covered with Matrigel (Millipore, USA).The upper chamber was added 0.1 mL cell suspension, while the lower chamber was filled with medium containing 20% FBS.After cultivated for 12 h, the cells under the lower surface of upper chamber was stained with 0.5% crystal violet (Beyotime, CHN) for 15 min at room temperature.Afterwards, an optical microscope was employed to observe and count the invasive cells.

Flow cytometry (FCM)
After 48 h transfection, the cells were trypsinised.After digestion, the cell pellet was collected by centrifugation at 1200 g for 5 min.Cells were resuspended and washed twice with pre-cooled phosphate-buffered saline (PBS).Subsequently, depending on the volume of the cell pellet, appropriate amounts of Annexin V-FITC and propidium iodide (PI) (Beyotime, CHN) were added and incubated for 3 and 10 min respectively.Finally, a flow cytometer (BD Biosciences, USA) was utilized to detect the cells apoptosis.

Xenograft assay
Four-week-old BALB/c nude mice were used for tumor transplantation.Mice were kept under sterile conditions with 12 h of light followed by 12 cycles of darkness.siRNA-Bmi-1 or RKIP were loaded into lentiviral vectors (), which were subsequently transfected into Weri-RB1 cells.2.5 × 10 6 transfected Weri-RB1 cells were resuspended in 100 μL PBS and injected into the axillae of the mice.Tumor size was measured with calipers as (length × width 2 )/2.After 20 days, mice were euthanized with carbon dioxide, tumors were removed and weighed.

Statistical analysis
All detection experiments are repeated 3 times, and the measurement data is expressed as mean ± SD.SPSS 21.0 software was used for statistical analysis.The two groups were compared by t-test, and the comparisons between three and more different groups were performed by the one-way analysis of variance LSD method.The difference was statistically significant when P < 0.05.

Knockdown of Bmi-1 restrains cell proliferation, migration and invasion, and increases cell apoptosis
To explore the roles of Bmi-1, the expression of Bmi-1 in retinoblastoma cell lines using RT-qPCR and Western blot.As expected, the expression of Bmi-1 was overexpressed in retinoblastoma cell lines Y79, SO-RB50 and Weri-RB1 than that of normal retinal vascular endothelial cell line ACBRI-181 (Fig. 1A,B).
To investigate the effect of Bmi-1 on tumor progression, siRNA-Bmi-1 was used to knockdown Bmi-1 in SO-RB50 and Weri-RB1 cells.The knockout efficiency was measured by RT-qPCR and Western blot (Supplementary Fig. 1A,B).MTT assay were used to measure the cell proliferation.MTT assay revealed that knockdown of Bmi-1 suppressed cell proliferation compared with control group at 24 h, 48 h and 72 h (Fig. 1C).Wound healing and Transwell assays were applied to detect the migration and invasion abilities.The results demonstrated that cell migration and invasion was reduced when silence of Bmi-1 in comparison with control (Fig. 1D,E).Cell apoptosis was measured using FCM.The results demonstrated that knockdown of Bmi-1 increased cell apoptosis (Fig. 1F).These findings revealed that inhibition of Bmi-1 suppressed cell proliferation, migration and invasion, and increased cell apoptosis in retinoblastoma.

Overexpression of Bmi-1 promotes cell proliferation, migration and invasion, and inhibits cell apoptosis
Bmi-1 was upregulated by transfecting overexpressed plasmid in Y79 and SO-RB50 cells, and the transfection efficiency was also detected by RT-qPCR and Western blot (Supplementary Fig. 2A,B).MTT assay indicated that cell proliferation was improved by overexpressing Bmi-1 at 26 h, 48 h and 72 h (Fig. 2A).Wound healing and Transwell assays revealed that overexpression of Bmi-1 increased cell migratory and invasive capacities (Fig. 2B,C).Overexpression of Bmi-1 inhibited cell apoptosis (Fig. 3D).These findings revealed that Bmi-1 increased cell proliferation, migration and invasion, and suppressed cell apoptosis in retinoblastoma.

Overexpression of RKIP suppresses cell proliferation, migration and invasion, increases cell apoptosis
Previous studies demonstrated that RKIP was a downstream gene of Bmi-1 18 .The expression of RKIP was calculated in retinoblastoma cells using RT-qPCR and western blot.As expected, the expression of RKIP was lower in Y79, SO-RB50 and Weri-RB1 cells than that of normal retinal vascular endothelial cell ACBRI-181 (Fig. 3A,B).To explore the roles of RKIP in retinoblastoma, the expression of RKIP was conducted to upregulate in SO-RB50 and Weri-RB1 cells, and the transfection efficiency was measured using RT-qPCR and western blot (Supplementary Fig. 3A,B).As expected, cell proliferation, migration and invasion were inhibited after overexpressing RKIP in SO-RB50 and Weri-RB1 cells (Fig. 3C-E).Overexpression RKIP increased cell apoptosis (Fig. 3F).All the findings revealed that overexpression of RKIP suppressed cell proliferation, migration and invasion, and increased cell apoptosis in retinoblastoma.

RKIP knockdown suppresses the functions of Bmi-1 knockdown on retinoblastoma cells
To investigate the effect of RKIP on tumor progression, siRNA-RKIP was used to knockdown RKIP in SO-RB50 and Weri-RB1 cells.The knockout efficiency was measured by RT-qPCR and Western blot (Supplementary Fig. 4A,B).siRNA-RKIP accelerated cell proliferation and reversed the inhibitory effect of siRNA-Bmi-1 on cell proliferation at 24 h, 48 h, and 72 h (Fig. 4A).Moreover, siRNA-RKIP promoted cell migration and invasion, and reversed the inhibition of siRNA-Bmi-1 on cell migration and invasion (Fig. 4B,C).Furthermore, siRNA-RKIP suppressed cell apoptosis and inhibited the pro-apoptotic effect of siRNA-Bmi-1 (Fig. 4D).The findings revealed that RKIP knockdown suppressed the role of Bmi-1 knockdown in retinoblastoma cells.

RKIP increases the functions of Bmi-1 knockdown on retinoblastoma cells
In addition, the expression of RKIP was upregulated after Bmi-1 knockdown in SO-RB50 and Weri-RB1 cells (Fig. 5A).On the contrary, RKIP was downregulated after overexpressing Bmi-1 (Fig. 5B).The cell proliferation of SO-RB50 and Weri-RB1 cells co-transfected with RKIP and siRNA-Bmi-1 at 24 h, 48 h and 72 h were decreased compared with cells only transfected siRNA-Bmi-1 or RKIP (Fig. 5C).Similarly, the migration and invasion ability of cells co-transfected with RKIP and siRNA-Bmi-1were lowest in all groups (Fig. 5D,E).The  www.nature.com/scientificreports/apoptosis of cells co-transfected with RKIP and siRNA-Bmi-1 were highest in all groups (Fig. 5F).All the findings revealed the functions of Bmi-1/RKIP axis in retinoblastoma tumorigenesis.

Downregulation of Bmi-1 suppresses xenograft tumor growth by regulating RKIP in vivo
To detect whether Bmi-1 and RKIP affect retinoblastoma tumorigenesis in vivo, nude mice were injected with Weri-RB1 cells transfected with siRNA-Bmi-1 alone or co-transfected siRNA-Bmi-1 with RKIP.The volume of the subcutaneous transplanted tumor was measured every 5 days, starting on day 5 after injection.Tumor growth was significantly attenuated after subcutaneous injection of siRNA-Bmi-1.What's more, the volume of transplanted tumor of mice injected with siRNA-Bmi-1 and RKIP were more downregulated relative to those only injected with siRNA-Bmi-1 (Fig. 6A).At day 20, mice were euthanized and the weighed and volume of tumors were calculated.The volume and weight of transplanted tumors were significantly suppressed in mice injected with siRNA-Bmi-1 compared to controls.Moreover, the volume and weight of transplanted tumors were smaller in mice injected with siRNA-Bmi-1 and RKIP than in mice injected with siRNA-Bmi-1 only (Fig. 6B,C).Bmi-1 expression was low in tumor tissues transfected with siRNA-Bmi-1 and in tumor tissues co-transfected with siRNA-Bmi-1 and RKIP (Fig. 6D).RKIP expression was high in tumor tissues transfected with siRNA-Bmi-1 and in tumor tissues co-transfected with siRNA-Bmi-1 and RKIP (Fig. 6D).These results suggested that downregulation of Bmi-1 inhibited tumor growth in vivo by regulating RKIP.

Discussion
Retinoblastoma is a common ocular malignant tumor in infants and young children.The global incidence of retinoblastoma is about 1/15,000, with about 9000 new cases per year 19 .The mortality rate of retinoblastoma was 40-70% in Asia and Africa, and 3-5% in developed countries in Europe and United States 20 .Retinoblastoma seriously endangers the eyesight and life of children, and brings great pain to children and their families.Therefore, it is of great significance to improve the diagnosis and treatment of retinoblastoma.
Bmi-1 induces the malignant transformation, which has been reported to be associated with tumor size, clinical stage and prognosis of gastric cancer 14,21 .Similarly, the modulation of BMI-1 leads to DNA damage, M phase cell cycle arrest, chromosome scattering and cell death in glioma 22 .What's more, BMI-1 expression is increased in early oral carcinogenesis and is possibly associated with the occurrence of dysplastic changes 23 .

Figure 6 .
Figure 6.Downregulation of Bmi-1 suppresses xenograft tumor growth by regulating RKIP in vivo.(A)Tumor growth was significantly attenuated after subcutaneous injection of siRNA-Bmi-1, and the volume of transplanted tumor of mice injected with siRNA-Bmi-1 and RKIP were more downregulated relative to those only injected with siRNA-Bmi-1.(B,C) The volume and weight of transplanted tumors were significantly suppressed in mice injected with siRNA-Bmi-1 compared to controls.Moreover, the volume and weight of transplanted tumors were more smaller in mice injected with siRNA-Bmi-1 and RKIP than in mice injected with siRNA-Bmi-1 only.(D) Bmi-1 expression was low, and RKIP expression was high in tumor tissues transfected with siRNA-Bmi-1 and in tumor tissues co-transfected with siRNA-Bmi-1 and RKIP.*P < 0.05, **P < 0.01, ***P < 0.001, compared to the Mock group; ## P < 0.01, ### P < 0.001, compared to the siBmi-1+RKIP group.